Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5' untranslated region

Abstract

A reverse-transcription polymerase chain reaction (RT-PCR) was developed to differentiate the bovine diarrhea virus (BVDV) from other pestiviruses, and to determine the genotype of the BVDV isolates. For this purpose, primer pairs were selected in the 5' untranslated region (5'UTR). The primers BE and B2 were located in highly conserved regions and were pestivirus-specific. Two primer pairs named B3B4 and B5B6 were specific of BVDV genotypes I and II, respectively. With this technique, an amplification product of the expected size was obtained with either the B3B4 or the B5B6 primer pairs for the 107 BVDV isolates tested but not for BDV or CSFV. For some isolates that were grouped in the genotype II, sequence analysis of the PCR fragments confirmed their classification into this genotype.

Fig. 1

Agarose gel electrophoresis of RT-PCR amplification products derived from BVDV NADL (1), New York (2), UVR420 (3), BDV (4), CSFV (5). M: Molecular weight marker XIV (Boehringer). (A) Primers B_EB₂, (B) Primers B₃B₄ and B₅B₆.

Fig. 2

Southern blot hybridization of pestivirus-specific amplification DNA fragments from BVDV NADL (1), New York (2), UVR420 (3), BDV (4), CSFV (5). M: Molecular weight marker 6 DIG (Boehringer). (A) B₄DIG Probe, (B) B₆DIG Probe.

Fig. 3

Alignment of nucleotide sequences from the 5′UTR of BVDV types I and II isolates. The sequences are flanked by primers B₁ and B₂. Genotype II sequences are presented above the horizontal line and genotype I below. Sequence of the B4 and B6 oligonucleotides is boxed.

Fig. 4

Phylogenetic tree of selected BVDV field isolates. A portion of the 5′UTR corresponding to BVDV-NADL base positions 206–374 was used. The horizontal branch lenghts are proportional to the similarity between the sequences.